1. Field of the Invention
The present invention relates to mutant strains of Pseudomonas putida and their use in the detoxification of industrial and/or toxic waste products.
2. Description of the Background
Many organic compounds synthesized from petroleum and other chemicals are highly toxic and recalcitrant. Herbicides, insecticides, chlorosubstituted dioxins and PCBs are known biohazardous compounds. Therefore, environmental protection efforts have been focussed on the development of more effective processes for the treatment of these toxic wastes.
Microorganisms used to process these hazardous wastes are likely to be exposed to a wide range of waste concentrations. Thus, this is one of the first problem that must be faced when addressing biological waste treatment issues. Above a certain threshold, toxic chemicals can kill a community of microorganism. Therefore, it is intrinsically important to confer a higher tolerance for waste chemicals, as pointed out by Timmis et al. ((1994) Annu Rev Microbiol 48:525–557). Soil bacteria has a wide range of metabolic abilities which make it a useful tool for mineralization of toxic compounds (reviewed by Chaudhry and Chapalamadugu, ((1991) Microbiol Rev 55: 59–79); Esteve-Nunez et al., ((2001) Microbiol Mol Biol Rev 65: 335–352); Kertesz and Wietek, ((2001) Appl Microbiol Biotechnol 57:460–466); Leahy and Colwell, ((1990) Microbiol Rev 54:305–315); Timmis et al., ((1994) Annu Rev Microbiol 48:525–557); Van der Meer et al., ((1992) Microbiol Rev 56:677–694)).
Pseudomonas is a major aerobic microorganism which can mineralize various organic compounds in environment. The greatest number of strains shown to be tolerant to organic chemicals belong to this genus (Inoue and Horikoshi, ((1989) Nature, 388:264–266); Kim et al., ((1998) Pseudomonas putida GM73 J Bacteriol 180:3692–3696); Ramos et al., ((1998) J Bacteriol 180:3323–3329); Weber et al., ((1993) Appl Environ Microbiol 59:3502–3504)). A single species of Pseudomonas as well as Comamonas is capable of growth on more than 80 organic compounds as a sole source of carbon and energy (reviewed by Palleroni, (1992) Introduction to the family Pseudomonadaceae. In the prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. Vol. 3 Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K.-H. (eds.) Springer-Verlag, New York. pp. 3071–3085). The pathways for metabolizing those compounds are highly specific and complex. Most of the enzymes in these catabolic pathways are known to be inducible (Chen et al., (1998) Res Communs Biochem and Cell & Mol Biol 2: 253–274). The discovery of microorganisms capable of tolerating to, or growing on, high concentrations of organic solvents provides a potentially interesting avenue for development of genetically engineered organisms for treating hazardous wastes. Solvent-resistance is inheritable and can be generated by random mutagenesis, and thus appears to be a useful phenotype for waste processing (reviewed by Timmis et al., (1994) Annu Rev Microbiol 48:525–557). Therefore, development of genetically enhanced microorganisms, through the use of genetic engineering, is important for developing better biological waste processing technologies.
In previous work (Hesketh and Ochi, ((1997) J Antibiot 50:532–535); Hosoya et al., ((1998) Antimirob Agents Chemother 42:204–2047); Hu and Ochi, ((2001) Appl Environ Microbiol 67:1885–1892)), we have demonstrated that the secondary metabolism of the genera Streptomyces and Bacillus (i.e. antibiotic production) could be activated or enhanced dramatically by introducing a mutation that confers resistance to streptomycin, gentamicin or rifampicin. These mutants have a mutation in the gene which encodes the ribosomal protein S12 (in Str mutants) or the RNA polymerase β-subunit (in Rif mutants) (Hu et al., ((2002) J Bacteriol 184: 3984–3991); Inaoka et al., ((2001) J Bacteriol 183:4958–4963); Ochi et al., ((1997) Mol Gen Genet 256:488–498); Shima et al., ((1996) J Bacterial 178:7276–7284); Xu et al., (2002) Mol Genet Genom, in press)).
In view of the damaging effects that toxic and industrial wastes pose to the environment and human health there remains a need for improved ways to detoxify these wastes.